This invention relates to an apparatus for separating and detecting substances relative to a living body such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid), etc. and in particular to a fluorescence detection type electrophoretic apparatus for analyzing the structure such as a base sequence in these substances by detecting fluorescence.
Heretofore the method, by which a substance to be examined is labeled with radioactive phosphorus (.sup.32 P), is utilized widely for determining the sequence of bases constituting a substance such as DNA (Tampakushitsu.Kakusan. Kohso (Protein.Nucleic Acid.Enzyme) Vol. 23, No. 3, p. 182 (1978)). By this method utilizing a radioactive label, one end of the substance such as DNA, etc., whose structure is to be determined, is labeled with .sup.32 P and after that, the substance is cut by provoking particular chemical reactions at certain positions of the bases by using a restriction enzyme. By controlling the rate of this chemical reaction, it is possible to produce fragments having .sup.32 P at the end, which have various lengths and each of which has a specified base at its severed end. They are separated by electrophoresis and detected by means of photographic plates, etc. This method has a disadvantage that a radioactive substance must be used and therefore a method, by which a substance to be examined is labeled with phosphor instead of radioactive substance and detected optically, is studied. In the case where phosphor is used for labeling, important problems for increasing detection sensitivity to fluorescence are how background noise can be reduced and how DNA etc., to which phosphor is added, are excited with a high efficiency.
In order to excite DNA fragments, to which phosphor is added, with light and to detect fluorescence thus emitted, the method as illustrated in FIGS. 1A and 1B is used. That is, light emitted from an excitation light source 6A passes a glass plate 1A and is projected into the gel portion 3. Further it passes another glass plate 1B and goes out into the exterior. If there exists a DNA fragment 21 in the path of the excitation light in the gel portion 3, the fragment is excited by the light and emits fluorescence, which is detected by a detector 7A. The output of the fluorescence detector 7A is treated by a data processing device 9, after having been amplified by an amplifier 8, and finally is displayed or printed out by an output device 10.
However, for this method, it is necessary to provide each of a plurality of electrophoretic paths with a light source or to divide a light beam emanating from one light source by means of mirrors, etc. to irradiate each fragment. Accordingly it has a disadvantage that the quantity of excitation light per fragment is reduced or the apparatus has a complicated structure. Further it has another disadvantage that, apart from fluorescence emitted by DNA fragments, to which phosphor is added, reflected light and fluorescence due to the glass plates holding electrophoretic gel give rise to background noise, which prevents measurements with a high sensitivity.